This invention relates to methods and compositions for controlling populations of Hymenopteran insect pests in the Formicidae (ant) family using a novel Bacillus thuringiensis (xe2x80x9cPTxe2x80x9d) toxin and preparation. In particular, the invention relates to effective methods of controlling populations of various fire ants of the family Solenopsis using a BT toxin effective in killing fire ants, and a novel strain of BT producing such toxin.
Without limiting the scope of the invention, its background is described in connection with uses of Bacillus thuringiensis toxins as fire ant biocidal agents as an example. The imported fire ant, Solenopsis invicta, is an introduced species that arrived in Mobile, Ala. from South America around the 1930s. The imported fire ant has spread rapidly across the southern United States and continues to expand into areas of North America with mild climates and adequate moisture and food.
The imported fire ant is an agriculturally and medically important pest harmful to domestic animals, wildlife and humans. Fire ants particularly threaten obligate ground dwelling species and young animals of all species. The imported fire ant is thought to be responsible for the decline of several native species. Proliferation of the imported fire ant has been largely unchecked due to the absence of predators, pathogens and parasites that control its numbers in its native environment. The fire ant typically causes painful stings in humans and more severe reactions may occur in allergic individuals. High densities of fire ants have been responsible for damage to roads, pastures, and electrical and mechanical equipment. The recent appearance of multiple queen colonies has made control of fire ant populations even more difficult. An effective compound and method for control of fire ant populations that is safe to users and consumers is urgently needed.
Past attempts at fire ant control have involved the highly toxic chlorinated hydrocarbons heptachlor, dieldrin, and Mirex. The EPA has outlawed the use of these highly toxic albeit relatively effective compounds for all but exceptional applications. The most commonly used modern control methods used include the chemicals hydramethylnon, avermectin, and the synthetic insect growth regulator fenoxycarb. These compounds must be regularly reapplied and have not been able to significantly impact severe fire ant infestations or to control further spread. The search for control methods is now being conducted in the areas of sterile insect release and the introduction of natural enemies, both of which are only potential solutions with uncertain outcomes.
Bacillus thuringiensis (xe2x80x9cBTxe2x80x9d) is the genus and species of a large number of strains of gram-positive, spore-forming bacteria which, under certain conditions, form a parasporal crystal comprised of insecticidal protein toxin (Bulla, et al., Crit. Rev. Microbiol., 8:147-204, (1980); Hxc3x6fte and Whitely, Microbiol Rev (1989) 53:242, (1989). The toxin itself is a glycoprotein product of cry genes (xe2x80x9cCryxe2x80x9d is used to denote the protein; xe2x80x9ccryxe2x80x9d is used to denote the gene) as described by Hxc3x6fte (Id.). Because the effects of the various Cry proteins are mediated by binding to unique receptors, the species specificity of a given BT toxin is typically quite limited as exemplified by the original classification proposed by Hxc3x6fte (Id.): CryI (Lepidopteran specific); CryII (Lepidopteran and Dipteran specific); CryIII (Coleoptera specific); and CryIV (Diptera specific). The BT toxins function in the brush border of insect midgut epithelial cells and, although highly insecticidal to certain insects, are non-toxic to other organisms lacking toxin binding receptors (Gill, S. S. et al. Ann. Rev. Entomol (1992) 37:615).
Keen interest in BT toxins over the last 30 years has resulted in the isolation of more than 100 different BT crystal protein genes and the development of bioinsecticides for the control of insect species in the orders Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Orthoptera and Mallophaga and against nematodes, mites and protozoa (Schnepf et al., Microbiol. Mol. Biol. Rev. (1998) 62(3):775). BT toxin in various forms now accounts for 90% of the world sales of non-chemical insecticides.
U.S. Pat. Nos. 5,260,058, 5,268,297, 5,596,071 and 5,824,792, disclose processes and compositions for controlling pharoah ants (Monomorium pharaonis) using toxin containing bacterial cells of various BT strains. Although these toxins are alleged to be effective against all Hymenoptera and all ants, no testing beyond the pharoah ant was performed. Further, the protoxins of this invention were much larger (120-140 kD) than that described herein.
Prior to this invention, no BT toxins have been reported that are known to be effective in controlling fire ants, nor are any BT toxins commercially available for this indication. What is needed is a biocidal composition that is effective in controlling populations of the fire ant, yet is not toxic to non-insect organisms. Such a biocide would have broad applicability including the agricultural, domestic, environmental and biomedical arenas. For this reason a BT toxin effective in killing fire ants would be particularly desirable.
Fire ants are omnivorous, although a large portion of their diet comprises invertebrates which the fire ants sting and kill. They also feed on dead animal and plant tissues, seeds, developing and ripe fruits, and are attracted to honeydew and sap flows. They are attracted to sugars, certain amino acids, ions in solution, and to some oils containing polyunsaturated fatty acids in these food sources.
Worker ants can only consume liquid foods, and nearly half of the resources that are returned to the nest are in the form of liquids. Liquids consumed and stored by the foraging workers are fed to other workers through trophallaxis. Once the worker arrives back at the colony, the oils are slowly transferred to nurses and from the nurses to larvae. Soluble sugars in addition to some soluble protein and amino acid mixtures are strongly attractive and encourage trophallaxis among workers. This both dilutes the solution and reduces the speed of movement of these nutrients to larvae.
Undissolved solids greater than 0.88 microns are screened from the liquid in the pharynx of the worker fire ant and cannot be ingested by the worker ants. The solids accumulate in the buccal region as pellets and are later expelled to feed fourth-instar stage larvae, which are able to consume particles as large as 45 microns. Because solid food may be used by the mature larvae but not the workers, solids move from the field to these larvae more quickly and directly than liquid foods. After processing by the fourth instar larvae, previously solid foods can be utilized by the queen and young larvae through trophallaxis. As it is desirable that worker ants transport poisons back to the colony and distribute the poison throughout the colony, consideration to the favored foods of fire ants together with the physical size of the poison may influence the efficacy of treatment modalities.
The present invention provides a BT protein that is active against members of the Formicidae family, in particular the imported fire ant, Solenopsis invicta, and related species. Generally, the invention provides BT preparations that are effective in reducing populations of fire ants and related species. The invention further provides novel B. thuringiensis strains that produce toxins effective against fire ants and related species. Effectiveness is defined as the ability to reduce the numbers of ants within a local fire ant population, typically a mound-type community, by killing immature and/or mature individuals within the population who are exposed to the toxin. Exposure may take the form of ingestion of the subject toxin either directly in a bait formulation or as expressed in a food source or by trophallaxis.
This invention provides biologically pure cultures of an isolated BT bacterial cell having the identifying characteristics of strain UTD-001 (NRRL No. B-30356).
In an alternate embodiment, the invention further provides recombinant DNA derived from the BT strain having the identifying characteristics of UTD-001 and encoding BT Cry toxin proteins. These characteristics include an approximately 73 kD wild type Cry protoxin protein that is activated on proteolytic processing to an approximately 67 kD toxin, has toxic activity against fire ants, has activity between pH 5-7, an SDS-PAGE profile substantially as shown in FIG. 2, and an arbitrary-primed PCR product profile substantially as shown in FIG. 3.
The DNA encoding BT Cry toxins may be isolated and cloned as recombinant DNA by the methods known in the art such as for example in the standard cloning manual of Sambrook, et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 2nd ed. 1989). The cry gene from UTD-001 has been cloned, and the sequences of the invention are identified as follows:
The invention further provides vectors for the cloning and expression of recombinant DNA encoding BT Cry protoxins or toxins that are toxic to fire ants. According to methods known in the art, the vectors may be adapted for expression of the Cry protoxin or toxin protein in various host organisms as desired. The invention contemplates the use of these expression vectors to produce novel host organisms which are engineered to express the encoded proteins either transiently, upon induction, or constitutively. Host organisms include those that are merely used as factories for the production of large quantities of toxin which is then purified and formulated into a bait. Alternatively, host organisms include organisms engineered to both express the protein and to serve as a food source for fire ants. Host organisms may include prokaryotic cells and eukaryotic cells and organisms such as for example, yeast, fungi, plants or animals.
The invention further provides purified Cry protoxins and toxins and compositions of these proteins that are active against Hymenoptera, Formicidae, and especially Solenopsis. In one embodiment, the Cry protoxin disclosed herein is characterized by a molecular weight of about 73 kD by SDS-PAGE, and 72.9 kD by calculation from the conceptually translated protein. The protoxin is putatively cleaved at residue 55 (between EA) by papain, and at residue 57 (between LD) by trypsin to produce an active protein. Thus, the protoxin is reduced to a toxin of about 67 kD by proteolytic digestion. However, it is expected that this cleavage site is somewhat flexible resulting in small variations in size of the active toxin. The novel Cry toxin of the present invention may also be described as a Cry3A-like toxin based on its close homology to the Cry3A toxin of Btt.
In one such embodiment, the Cry toxin is toxic to Formicidae, including Solenopsis invicta, S. richteri, S. xyloni, S. geminatam, S. geminata (aka Atta geminata Fabricius or S. geminata Mayr, S. japonica (aka S. fugax var. japonica), S. saevissima, S. orbuloides, S. punctaticeps, a variety of Solenopsis species identified by number, and related fire ant species. The invention provides methods for reducing ant populations by compositions containing Cry toxin or protoxin produced by BT strains having the identifying characteristics of UTD-001.
In yet another embodiment of the present invention, a biocide is produced by (a) propagating B. thuringiensis microorganisms having the identifying characteristics of strains UTD-001 under conditions wherein Cry protoxin is produced; (b) purifying the Cry protoxin; and (c) formulating the purified Cry protoxin into a biocide effective against ants. The Cry protoxin may be pre-activated by proteolytic digestion prior to formulating it into a bait, or may be produced directly in toxin form by the host organism.
In a further embodiment, the invention provides methods for isolating and cloning the BT toxin receptor of the imported fire ant and for using such receptor to engineer toxins with greater potency against the fire ant and for avoiding any resistance which may develop subsequent to the application of the present invention to fire ant populations.